PROJECT SUMMARY Skeletal muscle possesses a remarkable ability to regenerate due to the presence of resident muscle stem cells (satellite cells) but fails to do so in old age. Recent evidence suggests that factors circulating in the blood of young animals can rejuvenate muscle regenerative capacity of aged muscle. Heterochronic parabiosis, in which young and aged animals are surgically attached to share circulation, provided evidence of putative ?anti- geronic? factors exist but the mechanisms by which circulating soluble factors mediate rejuvenating properties on muscle stem cells and their microenvironment have yet to be elucidated. Due to the complexity of in vivo parabiosis and the dynamic nature of blood-borne factors, reliable identification of these humoral factors remains a major hurdle. To overcome this challenge, Dr. Jang and his team will leverage advanced microfluidic technologies and cellular engineering approaches to build a 3D microfluidic co-culture circuit that can control mechanical and biochemical cues in the physiologically relevant 3D microenvironment. His team seeks to assess circulating factors that influence myogenesis using the young-cell-laden VMOC (yVMOC) and old-cell- laden VMOC (oVMOC) to create a first-of-its-kind microfluidic, ?parabiosis-on-a-chip (PBOC)? to replicate in vivo parabiosis with tunable systemic and organ-specific parameters. Dr. Jang?s team will use the PBOC system to test the mechanism by which oxidative stress-induced systemic inflammation alters MuSC niche during aging. They will cross-validate the in vitro results with a mouse model that lacks an important antioxidant enzyme, CuZn superoxide dismutase (SOD1). Specifically, their proposed experiments will validate the vascularized muscle-on-a-chip (VMOC) as a platform to study systemic regulation of muscle stem cell function in vitro, and then test the effect of systemic oxidative stress on muscle stem cell function in vitro using systemic cross-circulation in the heterochronic PBOC. Lastly, their method combines PBOC and in vivo model system to test the hypothesis that the rejuvenation effect of heterochronic parabiosis is dependent on reducing pro-inflammatory cytokines. Dr. Jang and his team?s integrative system will provide unprecedented capabilities to reconstitute, visualize, and analyze pathophysiological processes relevant to skeletal muscle regeneration, and allow them to delineate the crosstalk between muscle, extracellular matrix, and micro-vascular network. The successful outcomes of this project will demonstrate advanced approaches to the development of 3D muscle culture system that closely mimics native in vivo muscle microenvironments, address critical technical barriers of in vivo parabiosis to identifying key soluble factors in the circulation that regulate cell, tissue and organismal aging, and serve as a foundation for translating the basic research and technology to the clinics. More importantly, following validation, the experimental approach used in this proposal can be readily translated into human parabiosis studies, which will have major clinical implications.